Project Summary The genome contains numerous non-coding DNA and RNA elements that far outnumber protein- coding genes, presenting a drastically new perspective of the transcriptional circuits. Among noncoding DNA elements, enhancers are the DNA fragments that activate target gene promoter by acting on gene promoters, independent of their distance or orientation to target genes. Many enhancers are recently found to encode noncoding RNAs, giving rise to an emerging class of enhancer RNAs (eRNAs). The eRNA mechanisms of heart failure, particularly at its interface with eDNA and chromatin, are largely unknown. We recently discovered a cardiac-specific eRNA capable of protecting the heart against pathological hypertrophy and failure. This program will focus on new mouse genetic models to define the molecular function of this newly identified eRNA in controlling enhancer function, 3-dimensional chromatin looping, cardiac gene expression, and pathological hypertrophy. Given that RNAs can be chemically modified and delivered as a drug for therapy, the success of this program will lay down a foundation for designing new mechanism-based therapy. Aim 1: Determining the in vivo sufficiency of an eRNA in cardioprotection. We will use transgenic and knock in/out technology of mouse genetics to define the cardioprotective role of this eRNA. Methods include CRISPR-enabled inducible and cardiomyocyte-specific eRNA modifications, transaortic constriction, histopathology, echocardiography, pressure-volume loop, and molecular marker studies. Aim 2: Defining how an eRNA interacts with its cognate eDNA in 3-dimensional chromatin looping and gene regulation. We will determine molecular interactions between an eRNA and its cognate eDNA in forming eRNA-eDNA hybrid duplex and in chromatin looping to target genes for transcription regulation under different pathophysiological conditions. Methods include mouse genetics, chromatin conformation capture, quantitative PCR, helicase assay, electric mobility shift assays, and DNA-RNA duplex immunoprecipitation. Aim 3: Defining eRNA-eDNA interactions in normal and diseased human hearts. We will use human heart tissues and iPS-derived cardiomyocytes to define the evolutionary conservation of eRNA-eDNA interactions and eDNA three-dimensional chromatin looping to target genes. Methods include ribosome profiling, in vitro transcription and translation, qPCR, chromatin fractionation and conformation capture, DNA-RNA duplex immunoprecipitation, and iPS-based technology.